Generally, analysis of a bio-related substance, such as cells or bacteria, is conducted by a technique whereby the bio-related substance is multiplied by culturing, and then an average value of a number of bio-related substances is determined. Meanwhile, a method for analyzing single bio-related substances has been proposed in recent years, and researches on cell response or suppression mechanism, cell-cell interaction, and stem cell differentiation are being conducted. As a result, it has been established that an averaged behavior of a number of bio-related substances greatly differs from the behavior of single bio-related substances. In this method, a single bio-related substance can be analyzed, so that not only a bio-related substance of which multiplication by culturing is difficult or a small number of bio-related substances can be analyzed, but also an early analysis can be performed by omitting the multiplying step for a culturable bio-related substance. Thus, it can be expected that 99.9% of bacteria that are currently considered difficult to culture, and a small number of patient-derived cells, such as stem cells, or bacteria, can be analyzed early, and therefore the analysis of single bio-related substances is becoming increasingly more important. In recent years, identification of circulating tumor cell (CTC), which is a cancer cell present in blood, and bacteria is also being conducted.
In order to analyze the single bio-related substances, a plurality of bio-related substances needs to be placed at single and independent positions. The “independent positions” herein refer to positions such that single bio-related substances can be identified in the device for analyzing the substances. The method for placing the bio-related substances at the single and independent positions may be largely categorized into methods utilizing fluid dynamics, methods utilizing external forces, such as electricity, light, magnetism, or ultrasound, and methods utilizing a surface treatment or chemical coupling, as discussed in the review paper of Non Patent Literature 1. It should be noted here that, when the bio-related substances are placed, the phenotype of the bio-related substances should not be altered. For example, when the bio-related substance is placed by surface treatment or chemical coupling, there is the possibility of chemical change or structure change in the bio-related substance, resulting in analyzing the behavior of a different bio-related substance. Further, because the method using external force is difficult to control, it may be most desirable to use a bio-related substance placing method based solely on fluid dynamics. In the following, the outline and problems of a report of the bio-related substance placing method based on fluid dynamics will be described.
The most general method for analyzing single bio-related substances involves enclosing a solution containing the bio-related substance between a slide glass and a cover glass and observing the solution. According to a method, degeneration or nonspecific adsorption of the bio-related substance is prevented by using a slide glass coated with an agarose gel or a blocking agent (such as BSA or Casein). In another method, the solution containing the bio-related substance is sent to a flow passageway of a microchip and observed in a stationery state. By either method, the bio-related substances can be placed at single and independent positions by adjusting the concentration of the solution containing the bio-related substances. For example, a case is considered where 10 μl of a solution containing bio-related substances is enclosed in a gap (0.025 mm) between a slide glass and a cover glass (20 mm×20 mm). When it is desired to place the bio-related substances at average intervals of 1 mm, the solution containing the bio-related substances at the rate of 4×104/ml (40/μl) may be prepared. When it is desired to place the bio-related substances at average intervals of 0.1 mm, the solution containing the bio-related substances at the rate of 4×106/ml (4000/μl) may be prepared. However, in the above method, except for the case where the bio-related substances are nonspecifically adsorbed on the slide glass or the cover glass and the like, the position of the bio-related substances are irregularly changed by Brownian motion or the motility of the bio-related substances themselves, for example. Thus, once the bio-related substance is out of the observation field of view, it is difficult to identify the same bio-related substance again. Further, because the intervals of the bio-related substances are average values after all, the bio-related substances are not always present at the independent positions and may be adjacent to another bio-related substance (in this case, the analysis device cannot determine that the bio-related substances are adjacent to each other). Another problem is that it is difficult to follow the movement of the bio-related substances. Further, only a minute amount of solution (in the present case, 10 μl) containing the bio-related substances can be used for analysis.
In another method, a microtiter plate is used. In this method, in a microtiter plate having a number of microwells of sizes ranging from fL (10−15 L) to pL (10−12 L), a concentration-adjusted solution containing the bio-related substances is dispensed such that, probabilistically, one bacterium enters one microcell. By designing the size of the microwells to be slightly greater than the size of the bio-related substances to be placed, entry of a plurality of bio-related substances into the same microcell can be prevented. A report has been made where single cells (several dozen μm) were introduced into 80 to 90% of microwells using this method. However, when the size is small such as on the order of several μm, as in the case of, e.g., bacteria, the application may become more difficult if the shape is not spherical but bar-like and the substances themselves have motility. When the gap between the microwells and the cover glass is filled with liquid, the bacteria having motility may possibly flow out. Further, in order to replace the solution in the microwells, large amounts of fluid and time are required due to poor substitution efficiency, making recovery of the bio-related substances difficult. In addition, only a minute amount of solution containing the bio-related substances can be used for analysis.
According to a method for solving the problem of being capable of analyzing only a minute amount of solution containing the bio-related substances, the flow passageway of a microchip is provided with a structural member for trapping the bio-related substances, and the bio-related substances are trapped as the solution containing the bio-related substances flows. In this method, only a necessary amount of the bio-related substances can be sent in a solution, eliminating the need to adjust the concentration of the solution containing the bio-related substances, and making it possible to use a very thin solution. In the following, the outline and problems will be described with regard to a report of a bio-related substance placing method based on this method.
In Non Patent Literature 2, as the structural member for trapping CD 34 cell of approximately 10 μm, a basket-shaped trapping structural member provided with slits of several μm that are smaller than the cell at three locations is used. A solution containing a bio-related substance is sent and passed through the slit portions, whereby the solution is introduced into the basket-shaped trapping structural member. If the basket-shaped trapping structural member does not have the slits, it becomes difficult to flush out air (air bubbles) present before the solution is sent, which is not practical. As much of the solution containing the bio-related substance can be introduced into the basket-shaped trapping structural member as the amount of the solution containing the bio-related substance discharged from the slit portions. At this time, the introduced bio-related substances cannot pass the slits, so that the substance is trapped by the basket-shaped trapping structural member. Because the basket-shaped trapping structural member is of a similar size to the CD 34 cell, the probability of a plurality of CD 34 cells entering the same trapping structural member is lowered, whereby single CD 34 cells can be trapped in most of the trapping structural members. By varying the width or number of the slits of the trapping structural member, the ratio of the solution containing the bio-related substance that can be introduced into the trapping structural member can be varied, whereby the bio-related substance trap rate is changed. In this case, there are the following four problems. The first is that, while the bio-related substance of a known size can be trapped by setting the slit width smaller than the size, it is necessary to fabricate the slit with smaller width as the size of the bio-related substance becomes smaller. For example, while the CD 34 cell is approximately 10 μm, the minor axis of bacteria is approximately 0.5 to 1.0 μm. Because the value of the slit width design value to which a maximum process error of the slit is added must be smaller than the size of the bio-related substance, a high process accuracy on the order of 0.1 μm is required, resulting in an increase in manufacturing cost. Further, when a narrow width of slit is used, the bio-related substance trap rate is decreased. The second problem is that, while the probability of a plurality of bio-related substances being trapped by the same trapping structural member is lowered by making the size of the trapping structural member similar to that of the bio-related substance so that single bio-related substances can be trapped in most of the trapping structural members, when the size or shape of the bio-related substance is diverse, a plurality of bio-related substances may be trapped in one trapping structural member. For example, the lengths of bacteria include the minor axis (approximately 0.2 to 1.0 μm) the major axis (approximately 1 to 10 μm), thus extending in a wide range. The third problem is that, because not all of the bio-related substances can be trapped by the trapping structural member and only the trapped bio-related substances are used as the object for analysis, important information may be missed, or the number of the bio-related substances cannot be quantified. The fourth problem is that, as the bio-related substance is continuously subjected to the force pulling it into the slit portion, stress may be caused in the bio-related substance, possibly resulting in a change in phenotype. While it appears that in Patent Literature 1, the trapping structural member does not have a slit, there is a gap of 2 μm in the Z direction, thus providing the same function as the slits in Non Patent Literature 2.
In Non Patent Literature 3, using a trapping structural member having a slit of approximately 0.8 μm, a single Escherichia coli is trapped at different trapping structural member positions. As opposed to Non Patent Literature 2 or Patent Literature 1, the trapping structural member is made sufficiently larger in size than the bio-related substance, and the probability of trapping the bio-related substance is lowered so that, as a result, single bio-related substances can be trapped in the same trapping structural member. However, there are the following problems. The first is that Escherichia coli of 0.8 μm or smaller is not trapped. Non Patent Literature 4 also reports that Escherichia coli can pass a flow passageway of a size one half its own minor axis, depending on the condition. In this case, even Escherichia coli of 1.6 μm may pass depending on the condition. The second problem is that not all of the bio-related substances can be trapped by the trapping structural member, and no analysis of the conditions is conducted. The third problem is that, as the bio-related substance is continuously subjected to the force pulling it toward the slit portion, stress may be caused in the bio-related substance, possibly resulting in a change in phenotype. The fourth problem is that, as discussed in Non Patent Literature 3, when the solution is sent at high speed, the bacteria may possibly be dissolved.